The biosensor based on the pyruvate oxidase modified conducting polymer for phosphate ions determinations

An enzymatic biosensor was fabricated by the covalent immobilization of pyruvate oxidase (PyO) onto the nano-particle comprised poly-5,2':5',2'-terthiophene-3'-carboxylic acid, poly-TTCA (nano-CP) layers on a glassy carbon electrode (GCE) for the amperometric detection of the phosphate ions. The direct electron transfer reaction of the immobilized PyO onto the nano-CP layers was investigated and the electron transfer rate constant was determined to be 0.65 s(-1). The electrochemically prepared nano-CP lowered the oxidation potential (+0.40 V versus Ag/AgCl) of an enzymatically generated H(2)O(2) by PyO in a phosphate solution. Experimental parameters affecting the sensitivity of the biosensors, such as amounts of the cofactors, the pH, the applied potential, and the temperature were optimized. A linear response for the detection of the phosphate ion was observed between 1.0 microM and 100 microM and the detection limit was determined to be about 0.3 microM. The response time of the biosensors was about 6s. The biosensor showed good selectivity towards other interfering anions. The long-term storage stability of the phosphate biosensor was studied and the sensor was applied in a human serum sample for the phosphate ions detection.     

Effect of chronic hypoxia on proliferation, apoptosis, and HSP70 expression in mouse bronchiolar epithelial cells

Heat shock proteins (HSPs) can be induced by various stresses and play an important role in cell cycle progression. HSP70 has been shown to act as an inhibitor of apoptosis. We studied HSP70 expression in bronchial epithelial cells of C57BL/6 mice and homozygous HPS70 knockout mice (hsp70.1-/-) exposed to chronic hypoxic stress. We also investigated changes in cellular proliferation and apoptosis in relation to HSP70. Lungs were removed from mice after a three-week period of exposure to 10 % O(2). Immunoblots for HSP70 and immunohistochemical staining for HSP70 and Ki-67 were performed. Apoptosis was assessed using the TUNEL assay. The three-week period of hypoxic stress did not change HSP70 levels in total lung tissue, but a significant reduction in HSP70 expression was observed in bronchiolar epithelial cells. In wild type mice, both HSP70 and Ki-67 expression were significantly reduced in bronchiolar epithelial cells. In homozygous HPS70 knockout mice (hsp70.1-/-), apoptosis of bronchiolar epithelial cells was significantly increased. Our results suggest that HSP70 may exert anti-apoptotic effects in mouse bronchiolar epithelial cells.     

Scalable production of adeno-associated virus type 2 vectors via suspension transfection

Vectors derived from adeno-associated virus type 2 (AAV2) are promising gene delivery vehicles, but it is still challenging to get the large number of recombinant adeno-associated virus (rAAV) particles required for large animal and clinical studies. Current transfection technology requires adherent cultures of HEK 293 cells that can only be expanded by preparing multiple culture plates. A single large-scale suspension culture could replace these multiple culture preparations, but there is currently no effective co-transfection scheme for generating rAAV from cells in suspension culture. Here, we weaned HEK 293 cells to suspension culture using hydrogel-coated six-well culture plates and established an efficient transfection strategy suitable for these cells. Then the cultures were gradually scaled up. We used linear polyethylenimine (PEI) to mediate transfection and obtained high transfection efficiencies ranging from 54% to 99%, thereby allowing efficient generation of rAAV vectors. Up to 10(13) rAAV particles and, more importantly, up to 10(11) infectious particles were generated from a 2-L bioreactor culture. The suspension-transfection strategy of this study facilitates the homogeneous preparation of rAAV at a large scale, and holds further potential as the basis for establishing a manufacturing process in a larger bioreactor.     

Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1)

We recently identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene induced in adventitial fibroblasts after arterial injury. Cthrc1 is a 30 kDa secreted protein that has the ability to inhibit collagen matrix synthesis. Cthrc1 is also glycosylated and retains a signal sequence consistent with the presence of Cthrc1 in the extracellular space. In injured arteries and skin wounds, we have found Cthrc1 expression to be associated with myofibroblasts and sites of collagen matrix deposition. Furthermore, we demonstrated that Cthrc1 inhibits collagen matrix deposition in vitro. Using in situ hybridization and immunohistochemistry, we characterized the expression domains of Cthrc1 during murine embryonic development and in postnatal tissues. In mouse embryos, Cthrc1 was expressed in the visceral endoderm, notochord, neural tube, developing kidney, and heart. Abundant expression of Cthrc1 was observed in the developing skeleton, i.e., in cartilage primordia, in growth plate cartilage with exclusion of the hypertrophic zone, in the bone matrix and periostium. Bones from adults showed expression of Cthrc1 only in the bone matrix and periostium while the articular cartilage lacked expression. Cthrc1 is typically expressed at epithelial-mesenchymal interfaces that include the epidermis and dermis, basal corneal epithelium, airway epithelium, esophagus epithelium, choroid plexus epithelium, and meninges. In the adult kidney, collecting ducts and distal tubuli expressed Cthrc1. Collectively, the sites of Cthrc1 expression overlap considerably with those reported for TGF-beta family members and interstitial collagens. The present study provides useful information towards the understanding of potential Cthrc1 functions.     

High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop March 7-9, 2007 Wellcome Trust Conference Centre, Hinxton, UK

The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled "High Performance Proteomics". It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics.     

Echinostoma ilocanum infection in Oddar Meanchey Province, Cambodia

Fecal examinations using the Kato Katz technique were performed on a total of 1,287 villagers (945 students and 342 general inhabitants) of Oddar Meanchey Province, Cambodia in May 2007 and November 2009. The overall intestinal helminth egg positive rate was 23.9%, and the most prevalent helminth species was hookworms (21.6%). Other helminth eggs detected included echinostomes (1.0%), Enterobius vermicularis (0.8%), small trematode eggs (0.7%), which may include Opisthorchis viverrini and Haplorchis spp., and Hymenolepis nana (0.4%). In order to recover adult echinostomes, we treated 2 patients with 10-15 mg/kg praziquantel and purged. Total 14 adult echinostomes, 1 and 13 worms from each patient, were collected. The echinostomes characteristically had 49-51 collar spines and 2 round or slightly lobated testes. They were identified as Echinostoma ilocanum (Garrison, 1908) Odhner, 1911. So far as literature are concerned, this is the first record on the discovery of human E. ilocanum infection in Cambodia.     

Isoegomaketone induces apoptosis through caspase-dependent and caspase-independent pathways in human DLD1 cells

Isoegomaketone (IK) is an essential oil component of Perilla frutescens (L.), but the mechanism by which IK induces apoptosis has never been studied. The purpose of this study was to elucidate the IK-induced apoptotic pathway in DLD1 human colon cancer cells. We observed that IK treatment over 24 h significantly inhibited cell viability in a dose-dependent manner. We also found that IK triggered cleavage of PARP. Moreover, IK treatment resulted in cleavage of caspase-8, -9, and -3 in a dose- and time-dependent manner. IK treatment also resulted in cleavage of Bid and translocation of Bax, and triggered the release of cytochrome c from the mitochondria to the cytoplasm. Furthermore, it resulted in the translocation of apoptosis inducing factor (AIF), a caspase-independent mitochondrial apoptosis factor, from the mitochondria into the nucleus. Overall, these results suggest that IK induces apoptosis through caspase-dependent and capase-independent pathways in DLD1 cells.     

Local kallikrein�Ckinin system is involved in podocyte apoptosis under diabetic conditions

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.